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Two academic teams have independently shown that targeting
the inflammasome reduces retinal damage in mouse models of age-related macular
degeneration.1,2 One study favors inhibiting inflammasome activation
to treat dry AMD, whereas the other suggests boosting it to treat wet AMD. The
approach for treating dry AMD has been licensed to iVeena Pharmaceuticals Inc., and the
academics focused on wet AMD are developing their own gene therapy.

Inflammasomes
are a family of cytosolic protein complexes that consist of three subunits: one
of several nod-like receptor proteins (NLRPs), the PYD
and CARD domain containing (PYCARD;
ASC)
protein, and caspase-1 (CASP1). In immune cells, activation of the inflammasome by
pathogens triggers release of the proinflammatory cytokines IL-1b and IL-18,
which then recruit effector cells to the site of tissue injury or infection as
part of the innate immune response.

The
inflammasome and its downstream effector cytokines have been implicated in
inflammation, cancer and metabolic disorders.3,4 However, until now
there had been no evidence that the complex plays a role in AMD and other eye
diseases.

Drusen are deposits of extracellular
proteins that form under the retina and are associated with risk for developing
AMD. Despite the correlation between AMD and large drusen deposits in the
macula, the underlying mechanism of drusen toxicity has remained unclear.

A
team of Irish researchers hypothesized that drusen might cause toxicity through
activation of the NLRP3 inflammasome because molecular aggregates, crystals and
deposits are known to activate the inflammasome in various other diseases.
These include cholesterol crystals in
atherosclerosis,5 islet amyloid
peptide aggregates in type 2 diabetes6 and uric acid crystals in gout.7

Moreover,
excess drusen is known to trigger cellular necrosis, a process that also
activates the NLRP3 inflammasome.8

To
test whether drusen activate inflammasome signaling in the eye, the team first
isolated drusen from donor eyes of deceased patients with AMD and added them to
cultured human peripheral blood mononuclear cells (PBMCs), which are immune cells
that enter the retina during AMD and express the NLRP3 inflammasome.

In those PBMCs, even low concentrations of drusen
triggered a significant increase in production of the two main inflammasome
effector cytokines-IL-1b
and IL-18-compared with no drusen (p<0.0001). In cultured murine PBMCs,
compared with wild-type PBMCs, Nlrp3 knockout led to decreased IL-1b production following addition of drusen.

Thus, drusen-induced upregulation of IL-1b and IL-18 in monocytes required
activation of the NLRP3 inflammasome.

Next, the researchers looked at the role of inflammasome
activation in the development and progression of AMD.

In a mouse model of wet AMD, knockout of Nlrp3 or Il-18 led to significantly greater
choroidal neovascularization (CNV) volume than that in wild-type controls (p<0.05),
suggesting that NLRP3 inflammasome activation and IL-18 expression both
protected against CNV. Intravitreal injection of an anti-IL-18 antibody also
significantly worsened CNV in the mice compared with no treatment (p=0.0368).

Taken together, the findings suggest inflammasome
activation in immune cells plays a protective role in wet AMD, whereby drusen
trigger the NLRP3 inflammasome to upregulate expression of IL-18. The cytokine,
in turn, reduces levels of proangiogenic VEGF.

The authors wrote that "a balance may exist whereby
a certain focal amount of drusen is tolerated because of its ability to induce IL-18,
which in turn may act as an anti-angiogenic effector." They concluded that
their findings suggest "strategies aimed at producing or delivering IL-18
to the eye may be beneficial in preventing the progression of CNV in the
context of wet AMD." However, "once a critical level of drusen
accumulates, its protective role is negated" and disease sets in, they
wrote.

Progression to wet AMD occurs in about 10% of cases of
the dry form, co-lead author Sarah Doyle told SciBX.

In the second paper, a group headed by
Jayakrishna Ambati, professor of physiology and vice chair of ophthalmology and
visual sciences at the University of Kentucky, found that
inflammasome activation worsened retinal damage in geographic atrophy, an
advanced form of dry AMD.

The
Kentucky researchers followed up on their own 2011 Nature paper that showed
that deficiency in the microRNA-processing enzyme dicer 1 ribonuclease type III (DICER1) led to accumulation of Alu RNA molecules in
the retinal pigment epithelium (RPE). The result was geographic atrophy and
retinal degeneration.9

Based
on those findings, Ambati and colleagues had originally planned to reduce
levels of toxic Alu RNA in the retina
using one of two approaches. "We reasoned we could either use Alu antisense therapy
to directly lower Alu RNA levels or use DICER1 gene therapy to
boost DICER1 levels, which would increase processing of Alu RNA and lower Alu RNA levels,"
said corresponding author Ambati.

"However,
we eventually abandoned both approaches. It proved too difficult to titrate the
antisense and gene therapies into the back of the eye without causing
dysregulation of RNA processing or unacceptable levels of inflammation,"
Ambati told SciBX.

The
team used a series of knockout mice and cell lines to successively eliminate
potential RNA-binding targets such as toll-like receptors (TLRs) and RNA-sensing proteins. For each knockout line, the
group compared levels of Alu RNA-mediated RPE
degeneration with those in wild-type controls, eliminating from further consideration
any protein whose knockout did not result in less degeneration than the
wild-type comparator.

Finally,
to determine whether the toxicity mechanism had any relevance to patients with
AMD, the researchers looked at whether human eyes with Alu RNA-associated
geographic atrophy (GA) also showed increased inflammasome signaling. Indeed, NLRP3
and IL-18 mRNA levels were significantly higher than those in normal eyes (p<0.05).

The
findings suggest inflammasome activation in RPE cells plays a destructive role
in dry AMD, whereby Alu RNA triggers the NLRP3
inflammasome to increase the activity of IL-18 and MYD88, which in turn leads
to RPE degeneration and GA.

"It is reasonable to foresee development of MyD88
inhibitors for prevention or treatment of GA," the authors concluded in
their paper in Cell.

Wet and dry
issue

Moving forward, the Trinity team will
develop an IL-18 therapy to enhance inflammasome activation in wet AMD, whereas
the Kentucky team will develop MYD88 inhibitors to block inflammasome
activation in dry AMD.

"Our
therapeutic strategy is based on the introduction of pro-IL-18 into the retina
using adeno-associated viruses (AAVs)," Trinity co-lead author Doyle told SciBX. Using pro-IL-18,
an inactive precursor form of IL-18, "is very important from a safety
point of view" because it will limit the activity of the proinflammatory
cytokine to only when and where it is needed.

"Our
AAV approach would be essentially self-regulating," added co-lead author
Campbell. "Pro-IL-18 is converted to its active form only in the presence
of the processing enzyme caspase-1, which is expressed in the retina when there
is a pathological insult such as excessive drusen prior to CNV development."

But
Ambati was skeptical about the prospects for any IL-18-based therapy. "Even
if IL-18 suppresses VEGF levels and is antiangiogenic, it would probably not be
a viable therapeutic strategy in any stage of AMD, given that our Cell paper shows
activation of the inflammasome and production of IL-18 is toxic to the retinal
pigment epithelium."

Campbell
countered that the findings of the Cell paper apply solely "to
end-stage dry AMD, not wet AMD, which is what our paper addresses. We are well
aware we are dealing with a proinflammatory cytokine here. Also, it must be
stressed that exceedingly small amounts of IL-18 are sufficient to have a
biochemical effect. We know the challenge moving forward will be determining a
level of IL-18 expression that is not damaging to the microenvironment of the
retina and yet can keep VEGF levels under control."

Ambati
suggested the Trinity group "make a vigorous evaluation of inflammasome
and IL-18 activation levels in the eyes of wet AMD patients before pursuing therapeutic
strategies along those lines."

It
would also be useful "to see if supplying exogenous IL-18 can suppress CNV
in an animal model of AMD," said Ryo Kubota, president, chairman and CEO
of Acucela Inc.

Acucela's
ACU-4429, an oral small
molecule visual cycle modulator, is in Phase IIa testing to treat dry AMD. The
compound "affects a process upstream of drusen formation and prevents its
accumulation," said Kubota.

Campbell said, "In the preclinical workup, we are
testing both the AAV approach as well as delivering recombinant IL-18 intravitreally.
In addition, we are exploring methods of systemic administration of IL-18 or
compounds that could induce expression of IL-18 locally in the eye."

At the moment, Campbell and colleagues are analyzing IL-18
and VEGF levels "not only in the eyes of deceased AMD patients, but we are
also trying to analyze the cytokines in vitreous and aqueous samples from
living patients with varying stages of AMD," he said.

Meanwhile, iVeena Pharmaceuticals, a biotech cofounded by
Ambati in 2006, has exclusively licensed the Cell findings from the University of Kentucky.
"The company's plan is to develop intraocular delivery of a MYD88 inhibitor
to treat geographic atrophy," said Ambati. "We will pursue that goal
along three parallel lines: small molecule screens, siRNA-based therapeutics
and peptide-based therapeutics."

Bruce Ksander, assistant professor of ophthalmology at Harvard Medical School,
said, "It is unclear to what extent dysfunction of Dicer and the
accumulation of Alu
RNA occurs in patients with geographic atrophy."

Ambati replied that "out of the few dozen eyes we've
studied from geographic atrophy patients, all have had elevated Alu RNA levels. So the phenomenon
appears uniform" across the patient population.

Ksander and Patricia D'Amore, professor of ophthalmology
and pathology at Harvard Medical School, have found that disruption of
lysosomes in RPE cells triggers activation of the NLRP3 inflammasome. Those
data have been submitted to an undisclosed peer-reviewed journal.

Ksander also is a scientist at the Schepens Eye Research
Institute. D'Amore is co-director of research and a senior scientist at the
Institute.

The Nature
Medicine findings are patented and available for licensing from Trinity
College.

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